Waterholes within the dryland Cooper Creek, Lake Eyre Basin, Australia, are connected only during floods and are typically isolated for long periods. Spatial changes in the macroinvertebrate assemblages of 15 of these waterholes belonging to four regions were explored and these changes were related to environmental aspects of the waterholes measured at four spatial scales: floodplain, waterhole, within waterhole and sample habitat. To explore temporal patterns, one region was sampled on four occasions differing in time since connection. Spatial patterns were characterised by ‘differentiation by distance’ whereby samples collected closer to each other in the landscape were more similar in assemblage composition than those collected further apart. Thus, there were significant differences between the assemblages of the four regions. Although there was a correlation between macroinvertebrate spatial patterns and a combination of local habitat, geomorphology and water chemistry attributes, it appears unlikely that these variables were responsible for the faunal differentiation by distance. Temporal variability was larger than spatial variability and temporal assemblage patterns were best explained by the ‘connectivity potential’ of waterholes, reflecting the position of individual waterholes within the broader channel network and long-term connectivity relationships, rather than the actual time since hydrological connection.